We are exploring the hypothesis that the Corticotropin Releasing Factor (CRF)/urocortin family of ligands and their cognate receptors play specific and critical roles in the modulation of adaptive responses to stress and other circumstances. In previous granting periods, this Project, with collaborators throughout the Program, characterized CRF, cloned 2 CRF receptors, CRFR1 and CRFR2 and identified urocortin (Ucn) 1, which had high affinity for both receptors. During the last period, we and the Sawchenko Project have found two additional ligands, Ucn 2 and 3, which are highly selective for CRFR2. We have focused on exploring the roles of the N-terminal, first extracellular domain (ECD1) of the CRFRs, which include critical binding sites for peptide agonists and antagonists. We have designed and expressed soluble forms of the ECD1's of CRFR1 and CRFR2beta with retained affinity for appropriate ligands and collaborated with Roland Riek, Salk Institute, to solve the structure of the CRFR2beta-ECD1 by NMR. In the next period, we will work closely with a new project headed by R. Riek, Core C and the Rivier Project to solve the structures of receptor ECD1- ligand complexes for CRFR1, CRFR2alpha and CRFR2beta as well as a novel soluble splice variant comprising mainly the ECD1 of R2alpha. The identification of a protein-protein interaction motif in the ECD1 spawned the testable hypothesis that the functional interaction that we had seen between CRFR2beta and erbB2 in cardiomyocytes may in part depend upon an interaction between the two receptors. Results with mice null for CRFR2 have suggested a variety of roles for this receptor and its ligands in the CNS, cardiovascular system and pancreatic islets. Mice deficient in each of the 3 urocortins have been developed in collaboration with the original Lee project and will be analyzed in this project with respect to the regulation of pituitary/adrenal and pancreatic hormones as well as the expression of CRF-related receptors and remaining ligands. Finally, we will examine the significance of Ucn 3 (and later, Ucn 2), shown to be produced in beta cells and to stimulate insulin and glucagon secretion, as local regulators of islet functions. In the context of the Program, progress in this Project should continue to yield information concerning the molecular nature, regulation and physiologic roles of ligands and receptors of the CRF family and provide insight regarding endocrine, metabolic and stress-related diseases.